Methods and kits for amplification of double stranded dna

ABSTRACT

Disclosed herein are devices and methods for amplifying double stranded DNA molecules. Methods for amplifying apoptotic cell-free DNA molecules can include performing end-repair and dA tailing of the cfDNA molecules, attachment of single-stranded hairpin adaptors to both ends of the end-repaired cfDNA molecules to produce adaptor-tagged, single-stranded, covalently closed DNA molecules, and amplification of the adaptor-tagged, single-stranded, covalently closed DNA molecules by a combination of rolling circle amplification and multiple displacement amplification using a PrimPol enzyme, a DNA polymerase with strand displacement activity and free nucleotides.

REFERENCE TO RELATED APPLICATIONS

This application is related to U.S. provisional application 62/589,074,filed November 21, 2017.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

None.

THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

None.

SEQUENCE LISTING

Appended hereto.

BACKGROUND

The last 10 years have seen an enormous progress in both theunderstanding of cancer disease as well as the development of targetedtherapies. An unprecedented number of new drugs has been approved forcancer treatment by the FDA and EMA over the course of the last 10years¹. Some of the most well-known drugs include Trastuzumab(Herceptin) for the treatment of breast cancer that targets the EGFR(HER2) receptor, Cetuximab (Erbitux) for the treatment of colorectaladenocarcinoma that targets the EGFR (HER1) receptor, Imatinib (Gleevec)for myeloproliferative disorders and CML targeting the Bcr-Abl fusionprotein, Sorafenib (Nexavar) for liver and kidney cancer targetingseveral tyrosine protein kinases, Crizotinib (Xalkori) for the treatmentof non-small cell lung carcinoma via inhibition of ALK-EML fusionproteins, or Vemurafenib (Zelboraf) for the treatment of melanoma viainhibition of the Braf V600E mutated kinase. The availability of a largeset of targeted therapies has created the need for efficient molecularprofiling of patients.

“Liquid biopsy” is a term coined to describe diagnostic procedures doneon nucleic acids in the blood or in other bodily fluids (e.g. urine orcerebrospinal fluid (CSF)) of patients ^(2, 3, 4). Cells dying byapoptosis or necrosis in a variety of diseases (cancer, myocardialinfarction, transplant rejection) release DNA from their fragmentedgenomes into the bloodstream. Also, DNA from a fetus can be detected inthe blood of the mother. A specific case are exosomes, 30-100 nmmicrovesicles, that next to DNA or RNA also contain proteins and lipidsfrom the originating cell^(5, 6, 7). These nucleic acids in the bloodcan be detected and analyzed using PCR-based methods, next generationsequencing (NGS), or array technologies. Data that have been generatedfrom analyzing liquid biopsies (e.g., nucleic acids obtained from bodilyfluids rather than from tumor masses) have shown the enormous potentialin this approach that could have a revolutionary impact on medicaldiagnosis, maybe similar only to the impact of the introduction ofmagnetic resonance imaging (MRI). Clinical applications that lookparticularly promising for the liquid biopsy approach are the diagnosisof chromosomal abnormalities in the fetus (in particular trisomy) byanalyzing the blood from the mother (called also non-invasive prenatalscreening (NIPT) based on cfDNA)^(8, 9, 10, 11, 12, 13, 14, 15), thediagnosis and monitoring of graft rejection in transplantation patients(DNA from donor tissue attacked by immune cells of the host can bedetected in the patient's blood)^(16, 17, 18, 19, 20), and diagnosis andmonitoring of cancer disease^(2, 4, 21, 22, 23, 24, 25). Areas withlimited data so far are other diseases with tissue necrosis orapoptosis, such as myocardial infarction²⁶. The use of “liquid biopsy”is most advanced in the detection of fetal chromosomal abnormalities (inparticular trisomies) where genomic diagnosis is challenging thetraditional combination of nuchal thickness measurement by ultrasoundand the triple test (AFP, hCG, and estriol)¹¹. However, the field withthe promise of highest medical impact is clearly oncology, where datagenerated during the last few years have shown that key cancer mutationscan principally be detected by liquid biopsy that mirror those presentin traditional tumor biopsies^(3, 27, 28).

Liquid biopsies may be superior to standard biopsies, as all parts of atumor and all metastases are potentially sampled. Recent data indicatethat in most cases analysis of circulating tumor DNA is faithfullyreflecting mutations found in all known metastases of a cancer, or iseven superior to such an approach (e.g. detecting mutations if standardbiopsies fail, or showing more mutations than the standard tissuebiopsies)²⁹, suggesting that sequencing circulating tumor DNA can give amuch more complete molecular picture of the systemic cancer disease thanstandard biopsies. Also, access to a patient's blood is unproblematic.Serial liquid biopsies can be easily taken to monitor cancer therapyeffects or to screen for reoccurrence of cancer as long as the volume ofblood needed for the respective analysis is small (e.g. a fewmilliliters). Sensitivity of the method may be superior for detectingcancer at a very early stage, e.g. in cases of reoccurrence of cancerdisease after curative surgery, or in a population-based screeningprogram. If liquid biopsy can improve early detection of tumors inpreventive screening programs will lead to a higher rate of cured cancerdisease, especially for tumor types where means of early detection andpreventive screenings are limited or non-existent.

Several studies of liquid biopsy approaches in cancer patients haverevealed that the success rate of this approach is related to the tumormass burden and tumor stage of a patient at the time of liquid biopsy,and the approach is not very successful in instances when tumor mass islow, because there are not so many tumor cells dying and releasing DNAinto the blood^(27, 30). Moreover, the approach works well in some tumortypes (e.g. colon carcinoma), but not in others (e.g. glioblastoma)presumably also due to sensitivity issues²⁷. Limitations due to verysmall amounts of cfDNA are likely more relevant in cases where cfDNA isto be analyzed by whole exome or whole genome sequencing as opposed tovery sensitive PCR-based approaches such as BEAMing³¹. Exome sequencingis advantageous to targeted PCR-based approaches, as practically thewhole exome-based single-nucleotide mutation landscape can be analyzedas opposed to only few and pre-known mutations that can be assessed bytargeted approaches. Therefore, exome sequencing has far more utilityfor early detection of cancer with high sensitivity, and of serialanalyses of the changing clonal landscape of a tumor followingtreatment. Often, in research laboratories that have only access tostandard library preparation and sequencing infrastructure, cell-freeDNA amounts of 100 ngs are required for library construction³². Withmore specialized approaches, exome sequencing has been done from cfDNAamounts of a minimum of 2.3 ng³³. Newman and colleagues have performedexome sequencing (“CAPP-seq”) from down to 7 ng cfDNA³⁴. DeMattos-Arruda used down to 22 ng of cfDNA input into libraryconstruction ³⁵.

A second problem that can diminish the detection power of liquid biopsyapproaches is the “contamination” of cell-free DNA with DNA coming fromunrelated processes (e.g. nucleated blood cells lysis during plasmaisolation). Cell-free DNA present in the blood plasma or other bodilyfluids (CSF, urine, ascites) can be broadly divided into the smallersize fragments (140-160 and 2-3×multiples of this) that originate fromapoptotic breakdown of genomic DNA inside a cell, and larger sizefragments that originate mainly from necrotic cell death (necrosis), butalso exosome shedding, and other less understood processes. DNAfragments of apoptotic origin can also be detected in healthy people andcan increase after sports or a cold for example. Current techniques forcell-free DNA analysis are composed of two principal types ofmethodologies: A) Next-generation sequencing (NGS): next generationexome sequencing^(33, 36), targeted (TAmseq³⁷; CAPPseq³⁴), FastSeqS³⁸,mFAST-SeqS³⁹, Safe-SeqS⁴⁰), or whole genome sequencing²⁸. Somecommercial kits have also been used in this (e.g. Thermo Fisher IonAmpliSeq Cancer Hotspot Panel v2). B) Digital PCR (BEAMing: beads,emulsions, amplification and magnetics^(31, 41, 42, 43, 44); digital PCRligation assay⁴⁵; emulsion based ddPCR with technology from RainDance orBio-Rad).

WO 2014/140,309 refers to methods for replicating, amplifying, andsequencing of nucleic acids using the thermostable, bifunctionalreplicase “TthPrimPol” from Thermus thermophilus HB27. It has been foundthat purified TthPrimPol displayed a strong DNA primase activity on asingle-stranded oligonucleotide in which a potential primase recognitionsequence (GTCC) is flanked by thymine residues. Such a tract ofpyrimidines has been shown to be the preferred template context forinitiation of the priming reaction by several viral, prokaryotic andeukaryotic RNA primases. It has been found that priming occurred only infront of the “TC” sequence, and that there was no priming opposite thepoly dT tracks. Further analysis of template sequence requirementsrevealed an effect of the nucleotide preceding the template initiationsite on TthPrimPol's primase activity—C is preferred over A, G or T.Even if TthPrimPol prefers CTC as template initiation site, it is ingeneral able to act as a primase on any sequence of the generic formXTC, where X stands for either of A, C, G, or T. The modest sequencerequirement forms an excellent basis for random priming of nearly allnatural templates.

Therefore, sensitivity and specificity limitations of current liquidbiopsy approaches are an area in need of technical improvement.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form a partof the specification, illustrate exemplary embodiments and, togetherwith the description, further serve to enable a person skilled in thepertinent art to make and use these embodiments and others that will beapparent to those skilled in the art. The invention will be moreparticularly described in conjunction with the following drawingswherein:

FIG. 1 shows cell-free DNA pretreatment to eliminate protruding ends,restore 5′ phosphates and 3′ hydroxyl groups, and add 3′ dA overhangs.

FIG. 2 shows a mechanism of an amplification kit for apoptotic cell-freeDNA.

FIG. 3 shows evidence that TruePrime technology requires the addition ofhairpin adaptors to efficiently amplify short DNA molecules (200 bp) byprimase-initiated multiple displacement amplification. “NTC” refers to“non-template control” (no DNA is added as input for the reaction).“HgDNA” stands for human genomic DNA.

FIG. 4 shows amplified DNAs (3 independent replicates) from FIG. 3(DNA 1) that were cut with EcoRl restriction enzyme obtaining theexpected unit size (200 bp from the original DNA fragment+36 bp from thehairpin adaptor).

FIGS. 5A and 5B show amplification yields (5A, upper part) depending onthe method used to initiate the amplification (TthPrimPol or randomprimers) and the result of the digestion with a restriction enzyme ofeach amplified DNA (5B, lower part).

FIG. 6 shows the amplification yields of short DNA molecules (from 50 to1250 bp), requiring the addition of hairpin adaptors, byprimase-initiated multiple displacement amplification.

FIG. 7 shows adaptors used to test the DNA primase activity ofTthPrimPol.

FIG. 8 shows TthPrimPol DNA primase activity observed in each hairpinadaptor.

FIG. 9 shows DNA amplification yields obtained from cfDNA samples from48 cancer patients, as well as the detailed protocol of theamplification process.

FIG. 10 shows the amplification yields in micrograms obtained fromdifferent amounts of cfDNA from a cancer patient.

FIG. 11 shows the high correlation of fragments separated by the hairpinadaptor sequence [SEQ ID NO:1] in respect to the length of the MinIONread (Oxford Nanopore).

FIG. 12 shows the genome coverage and copy number variant detection fromtwo amplified cfDNA samples from the same colon cancer patient.

FIG. 13 shows the coverage plot from the two amplified cfDNA samplesfrom the same colon cancer patient in different resolutions. This plotreflects the distribution of reads along chromosome 4 from two cfDNAsamples from the same colon cancer patient amplified with the methoddescribed herein. Whole genome sequencing was carried out and the readsobtained were aligned to the human reference genome. A very even andhomogeneous distribution of reads is observed in independentamplification reactions at different resolutions. The plot zooms in toshow how similar the distribution pattern in both samples is.

FIG. 14 shows the number of annotated and non-annotated variantsdetected in each of the three patients comparing the amplified andnon-amplified cfDNA samples in each case.

FIG. 15 shows the variant calling results obtained for the first patientat two different allele frequencies (1% and 35%) and the correlationbetween amplified and non-amplified cfDNA samples.

FIG. 16 shows the variant calling results obtained for the secondpatient at two different allele frequencies (1% and 35%) and thecorrelation between amplified and non-amplified cfDNA samples.

FIG. 17 shows the variant calling results obtained for the third patientat two different allele frequencies (1% and 35%) and the correlationbetween amplified and non-amplified cfDNA samples.

FIG. 18 shows an amino acid sequence for TthPrimPol [SEQ ID NO: 10].

SUMMARY

Analysis of cell-free DNA in oncology and other fields offers hugeopportunities to improve diagnosis and treatment in patients. A keyproblem is the difficulty to obtain results from biological fluids suchas plasma, urine or CSF samples with very low cell-free DNA content.Methods disclosed herein provide a solution for this issue by amplifyingshort double-stranded polynucleotide fragments, includingdouble-stranded DNA. This includes, without limitation those moleculesfound in cell-free DNA. Amplification is based on an amplificationtechnology called “primase-initiated multiple displacementamplification” (“PI-MDA”, also referred to as “TruePrime”) combined witha novel set of sample pretreatments that allows its efficientamplification by subsequent steps such as rolling circle DNAamplification. As used herein, “primase-initiated multiple displacementamplification” or “TruePrime” refers to a form of multiple displacementamplification (“MDA”) that uses a primase/polymerase to provide primersfor primer extension by a DNA polymerase. Typically, the polymerase hasa very strong displacement capacity and good fidelity of synthesis toavoid sequence changes during the process. One such polymerase is Phi29DNA polymerase. While the current gold standard MDA needs short piecesof DNA (“oligonucleotides”) to start off the amplification,primase-initiated multiple displacement amplification does not need anysynthetic random primers.

The presently disclosed methods address, among other problems, thisliquid biopsy sensitivity and specificity issue by an adaptedamplification of apoptotic cell-free DNA based on the PI-MDA technology(“TruePrime”), which is a method of DNA amplification by iterativepriming, copying and displacement steps.⁴⁶ TruePrime kits and protocolsare available commercially from Expedeon AG and its affiliates. Thisdisclosure provides a combination of existing TruePrime with the addedsteps of a sample pretreatment, comprising an end-repair, dA tailing,and the ligation of hairpin adaptors (see FIG. 1). Individually, thesesteps may be carried out by methods known in the art, including by useof the New England Biolabs NEBNext Ultra II DNA Library Prep Kit andsimilar products and methods, and/or for example: (a) for end repair T4polynucleotide kinase (PNK)+T4 DNA polymerase Klenow fragment and T4 DNApolymerase large fragment are commonly used (b) for dA tailing Taqpolymerase is commonly used. This novel combination of methods providesan efficient process for what would otherwise be unworkable orinefficient: the amplification of small fragments of DNA by traditionalTruePrime or other methods. The method steps may optionally be followedby the rolling circle DNA amplification method (see FIG. 2). Thus, weprovide a novel method to amplify short DNA molecules, e.g. but notlimited to, apoptotic cell-free DNA without ligation, allowing anincrease in the DNA available for any analytical technology, withsuperior sensitivity and specificity. While the methods disclosed hereinprovide particular advantage for short DNA fragments they also work wellwith DNA samples of any length.

DETAILED DESCRIPTION I. Introduction

PrimPol is an enzyme obtained from the thermophilic bacteria Thermusthermophilus. PrimPol combines two distinct and complementary activitiesin a single thermo-stable protein: primase and polymerase. Conventionalpolymerases require small stretches of nucleotides (primers) annealed toa template molecule to synthesize the complementary sequence. PrimPol,on the contrary, creates its own primer sequence, thereby offering fullynovel applications.

Moreover, PrimPol is able to copy both DNA and RNA. RNA reflects whatgenetic information is actually expressed in a cell, whereas DNA refersto the general genetic information present in every cell in the body andoften only reflects a predisposition of a person to develop a disease.The development of PrimPol will help to simplify technical aspects ofDNA and RNA amplification procedures.

PrimPol also shows a great tolerance to damaged DNA. DNA is subject tochemical modifications within the cells. Also during the processesnecessary to purify the genetic material, and storage of forensic andclinical samples (e.g. formalin-fixed paraffin-embedded tissues) triggersuch modifications. Chemical modifications have been shown to play anincreasingly important role in several biological processes, such asaging, neurodegenerative diseases, and cancer. Therefore, there is greatinterest to develop methods for interrogating damaged DNA in the contextof sequencing. Thus, an enzyme able to handle modified templates is ofparticular interest, since current amplification applications as well assecond and third generation sequencing technologies are not optimized touse damaged samples.

PrimPol is also suited to be used in different second and thirdgeneration sequencing technologies due to its ability to introduce avariety of substrate nucleotides (e.g. fluorescent nucleotides) into DNAand RNA template molecules.

Finally, PrimPol has a role in multiple displacement amplification (MDA)reactions, generating primers for its subsequent use by Phi29 DNApolymerase, thus making unnecessary the use of random synthetic primersand possibly resulting in a more uniform amplification of DNA.

II. Methods of Amplifying Linear Double Stranded Polynucleotides

Provided herein are, among other things, methods of amplifying linear,double stranded polynucleotides (e.g., DNA molecules) and, inparticular, apoptotic (mononucleosomal) cell-free DNA. Methods ofamplifying linear, double stranded DNA include attaching single-strandedhairpin adaptors to both ends of the DNA molecules to produce singlestranded, covalently closed DNA molecules and amplifying the covalentlyclosed DNA molecules by rolling circle amplification and multipledisplacement amplification, e.g., primase-initiated multipledisplacement amplification.

A. Linear Double Stranded DNA

Linear double stranded DNA for use in the amplification methodsdescribed herein can be provided from any source. This includes, forexample, DNA from eukaryotes, eubacteria, archaebacteria and viruses.Eukaryotic sources of DNA can include plants, animals, vertebrates,mammals, and humans. Microbial sources of DNA can include microbessourced from a microbiome of an individual or from the environment.

The linear double stranded DNA used in the amplification methodsdisclosed herein can be of any length. In certain embodiments, thelinear double stranded DNA has a length of no more than 5000nucleotides, no more than 1000 nucleotides, no more than 500 nucleotidesor no more than 200 nucleotides. In other embodiments the population oflinear double stranded DNA molecules to be amplified can have an averagelength of no more than 5000 nucleotides, no more than 1000 nucleotides,no more than 500 nucleotides, no more than 200 nucleotides, no more than100 nucleotides, no more than 50 nucleotides, no more than 20nucleotides, e.g., about 168 nucleotides. DNA molecules to be amplifiedcan include molecules having a length between about 100 and about 220nucleotides. The linear double stranded DNA can comprise fragmentedchromosomal DNA. Such DNA fragments may have a length greater than 5000nucleotides.

Nucleic acids are typically isolated from other components by isolationmethods well known in the art including, without limitation, capture onparticles having DNA or RNA binding activity, such as silica particles;polyethylene glycol precipitation and SPRI (Solid Phase ReversibleImmobilization) beads.

1. Cell-Free Polynucleotides

The linear double stranded DNA used in the methods disclosed herein cancomprise cell-free DNA (“cfDNA”). Cell-free DNA refers to DNA that isnot encapsulated inside a cell. Cell-free DNA can be apoptotic cell-freeDNA. (FIG. 1, “Apoptotic cell-free DNA”.) Apoptotic cfDNA refers to DNAreleased from dead or dying cells, e.g., through the apoptosis celldeath mechanism, in which the DNA is cut between nucleosomes, producingDNA fragments of 160-170 bp, or multiples thereof, when the cut in theDNA is not produced between every nucleosome. This includes DNA releasedfrom normal cells, e.g. having a germline genome. It also includes DNAreleased from cancer cells (e.g. malignant cells), also referred to ascirculating tumor DNA or “ctDNA”. This DNA typically carries somaticmutations associated with cancer e.g., in oncogenes or tumor suppressorgenes. Apoptotic cfDNA also includes fetal DNA in the maternalcirculation. Cell free DNA can be sourced from any of a number ofdifferent bodily fluids including, without limitation, blood, plasma,serum, CSF, urine, saliva, tear drops, milk, semen and synovial fluid.Apoptotic cfDNA typically has a size distribution with two peaks; afirst mononucleosomal peak between about 110 to about 230 nucleotides,with a mode of about 168 nucleotides, and a second, minor dinucleosomalpeak between about 240 to 440 nucleotides.

Cell-free DNA molecules can be prepared from bodily fluids, such asblood, by conventional methods. Commercial kits for this purpose areavailable from, e.g., Thermo Fisher Scientific (Waltham, Mass., USA),Active Motif (Carlsbad, Calif., USA) and Qiagen (North Rhine-Westphalia,Germany). In general, cells are removed from a sample, for example bycentrifugation. Silica particles, e.g. magnetic silica beads, are addedto the sample from which cells have been removed. The silica particlesbind the DNA. The particles are isolated from the supernatant, forexample by centrifugation and/or application of magnetic force.Supernatant is removed and the particles are washed. Cell-free DNA isisolated from the particles by dilution with ethanol.

2. Other Polynucleotides

In other embodiments, the double stranded DNA molecules comprisefragmented genomic DNA, for example, isolated from cells or doublestranded cDNA molecules produced from reverse transcription of RNAmolecules, such as mRNA, rRNA or tRNA molecules.

3. End-Repair

End-repair refers to a process of providing double stranded DNAmolecules having 3′ and/or 5′ single strand overhangs with either stickyends or blunt ends. End repair renders double stranded DNA moleculesmore suitable for attachment to polynucleotide adaptors. Attachment canbe by either sticky-end ligation or blunt-end ligation. “Sticky-endligation” refers to the ligation of two double-stranded polynucleotides,each of which has a 3′ overhang complementary to the other 3′ overhang.A sticky end can be, for example, a single nucleotide overhang, such as3′ dA and 3′ dT, or a longer sticky-end, such as an overhang produced byrestriction enzyme digestion. “Blunt-end ligation” refers to theligation of a double stranded polynucleotide to the blunt-end ofanother, double stranded polynucleotide.

In either case, modification of the polynucleotide typically initiallyinvolves blunt ending the molecules. Typically, this involves using apolymerase to fill in a 5′ overhang and a molecule having exonucleaseactivity to chew back a 3′ overhang. Blunt-ending can be performed usinga mixture of T4 polymerase and the DNA polymerase I klenow fragment. Theklenow fragment possesses 5′-3′ polymerase activity to fill in 5′overhangs and 3′-5′ exonuclease activity to remove 3′ overhangs. T4 DNApolymerase possesses a less efficient 5′→3′ polymerase activity and amore efficient 3′-5′ exonuclease activity. Mung bean nuclease also canbe used to eliminate 5′ and 3′ overhangs. T4 polynucleotide kinase (“T4PNK”) is used to phosphorylate the 5′ strand of a molecule anddephosphorylate the 3′ strand. Kits for performing blunt-ending of DNAmolecules are available from a variety of commercial sources includingThermo Fisher Scientific (Waltham, Mass., USA) and New England Biolabs(Ipswich, Mass., USA).

In certain embodiments, a blunt-ended polynucleotide can be dA-tailed bya process of adding a terminal 3′ deoxy adenosine nucleotide to a DNAmolecule. This action can be performed using Taq polymerase. (FIG. 1,“End-repaired cfDNA (3′-dA overhangs)”.)

Blunt-ended molecules or dA-tailed molecules can be used in the methodsdescribed herein by the attachment of single-stranded adaptors.

B. Attachment of Single-Stranded Adaptors

Polynucleotides that have been end-repaired and, optionally, dA-tailed,can be ligated to adaptors. Adaptors are polynucleotide moleculesadapted for attachment to target molecules. Typically, adaptors includenucleotide sequences for priming DNA strand extension. In certainmethods of this disclosure the adaptor is a hairpin adaptor. As usedherein, the term “hairpin adaptor” refers to a single stranded nucleicacid molecule (e.g., DNA) that includes a second region flanked by afirst and third region. The first and third regions have sufficientcomplementarity to hybridize with each other (e.g., 95%, 99%, or 100%complementary). The second region is not complementary to either thefirst or the third region. The adaptor can have a length of between 25and 100 nucleotides long. The second region can be, for example, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or fewer than 25 nucleotides long.As a consequence, a hairpin adaptor molecule can fold back on itselfforming a stem-and-loop structure comprising a double stranded end and asingle-stranded, internal segment that is not hybridized. Hairpinadaptors can include a 3′ dA overhang, or be blunt-ended, depending onthe application. See, e.g., FIG. 7 and FIG. 8.

Adaptors also typically include internal nucleotide sequences compatiblewith a DNA primase and/or primer extension. For example, the hairpinadaptor can include a primase/polymerase recognition sequence, e.g.,XTC, where X represents nucleotides a, T, G or C, for example, CTC, GTCor GTCC. The primase/polymerase recognition sequence can be located inthe loop (non-complementary) portion of the adapter. Which is recognizedby TthPrimPol. In other embodiments the hairpin adaptor comprises anamplification primer binding site.

End repaired, dA-tailed double-stranded polynucleotides can be attachedon one or both ends to hairpin adaptors. The end of the adaptor ispreferably compatible with the end of the double strandedpolynucleotide. So, for example, a dA-tailed double-strandpolynucleotide is attached to a dT tailed hairpin adaptor, or ablunt-ended double-stranded polynucleotide is attached to a blunt-endedhairpin adaptor. Attachment is typically performed with a DNA ligase,such as T4 DNA ligase. In certain embodiments hairpin adaptors have asequence as depicted in FIG. 7, i.e., [SEQ ID NO:1], [SEQ ID NO:2], [SEQID NO:3], or [SEQ ID NO:4].

The product of ligation between hairpin adaptors and double-strandedpolynucleotides is a single-stranded, covalently closed polynucleotide.See, e.g., FIG. 1 (“Adaptor ligation”). Such a polynucleotide can alsobe described as a single stranded, circular polynucleotide. As usedherein, the term “covalently closed DNA molecule” refers to a DNAmolecule having no free 5′ or 3′ end. Such molecules are also referredto as “circular DNA”. Because they have complementary internalsequences, such molecules can assume a “dumbbell” shape. Apolynucleotide insert flanked on one or both ends by hairpin adaptors isreferred to as a “adaptor-tagged polynucleotide”.

A collection of adaptor-tagged polynucleotides is referred to as a“nucleic acid library”. Typically, in the case of cfDNA, a nucleic acidlibrary comprising a population of end-repaired DNA molecules includepolynucleotide inserts having different nucleotide sequences.Single-stranded, covalently closed polynucleotides can be amplified bymethods disclosed herein.

C. Amplification

In certain embodiments, amplification of single-stranded, covalentlyclosed polynucleotides involves using a DNA-directed primase/polymerase,such as TthPrimPol; a DNA polymerase having strand displacementactivity, such as Phi29; and modified or unmodifieddeoxyribonucleotides. In combination, these reagents effect rollingcircle amplification primed by the primase/polymerase and extended bythe DNA polymerase. Furthermore, the combination of primase/polymeraseand DNA polymerase can effect multiple strand displacement amplificationthrough priming of amplified molecules with the primase/polymeraseand/or random oligonucleotide primers and primer extension by the DNApolymerase. Multiple strand displacement amplification produces abranched structure as DNA synthesis is primed and extended from manypositions in the amplified molecules.

Furthermore, amplification can be accomplished without the use ofoligonucleotide primer molecules by using hairpin adaptors comprisingone or more primase recognition sites together with a primase having DNApriming activity on single stranded DNA, such as TthPrimPol, and a DNApolymerase having strand displacement activity, such as Phi29 anddeoxyribonucleotide triphosphates. Using these reagents, a highlybranched structure is produced during multiple strand displacementamplification.

1. Primase/Polymerase

As used herein, the term “priming” refers to the generation of anoligonucleotide primer on a polynucleotide template.

For amplification of DNA, the primase/polymerase can be a DNA-directedprimase/polymerase, such as a PrimPol enzyme. Unlike most primases,PrimPol is uniquely capable of starting DNA chains with dNTPs. UsefulPrimPol enzymes include, among others, Thermus thermophilusprimase/polymerase (“TthPrimPol”) and human primase/polymerase(“hsPrimPol”).

Thermus thermophilus HB27 primase/polymerase is described, for example,in WO 2014/140309, published Sep. 18, 2014 (“Methods for amplificationand sequencing using thermostable TthPrimPol”). It has an amino acidsequence shown in FIG. 18 [SEQ ID NO: 10]. It bears Gene ID: NC_005835in the NCBI Entrez database, protein WP_011173100.1 TthPrimPol can beobtained commercially in kits from Expedeon (Cambridge, UK).

Human PrimPol is also known as MYP22; CCDC111 and Primpoll. It bearsGene ID: 201973 in the NCBI Entrez database.

The PrimPol can be a relative of any PrimPol described herein includingthe following: An allelic variant (a naturally occurring variation of agene), an artificial variant (a gene or protein comprising one or moregenetic modifications to a naturally occurring gene or protein whileretaining natural function), a homolog (a naturally occurring gene fromanother genus or species than the one defined, or a distinct gene in thesame strain or species that encodes for a protein having nearlyidentical folding and function); an ortholog (a homolog that occurs inanother genus or species from the one discussed) or a paralog (a homologthat occurs in the same strain or species as the one discussed, e.g., asa result of gene duplication). A PrimPol enzyme can have at least 80%,85%, 90%, 95%, 98% or 99% amino acid sequence homology with the proteinof SEQ ID NO: 10.

2. DNA Polymerase with Strand Displacement Activity

Amplification methods can employ a DNA polymerase with stranddisplacement activity, e.g., a polymerase with strong binding tosingle-stranded DNA e.g., in preference to double-stranded DNA. Stranddisplacement activity can be useful in displacing hybridized strands ofa DNA molecule while extending a primer position, for example, in theloop area of a hairpin structure.

DNA polymerases with strand displacement activity useful in methodsdisclosed herein include, for example, Phi29. Phi29 DNA polymerase canbe obtained commercially from, for example, New England Biolabs(Ipswich, MA, USA), ThermoFisher Scientific (Waltham, MA, USA andExpedeon (Cambridge, UK). Phi29 polymerase can generate DNA fragments upto 100 kb. The enzyme has a 3′-5′ exonuclease proofreading activity andprovides up to 1000-fold higher fidelity compared to Taq DNApolymerase-based methods. Phi29 polymerase can function on DNAcomprising secondary structures such as hairpin loops.

In another embodiment the DNA polymerase can be Bacillussteatothermophilus (Bst) polymerase.

3. Deoxyribonucleotide Triphosphates

Primer creation and primer extension can be accomplished by providingprimase/polymerase enzymes and DNA polymerases with deoxyribonucleotidesubstrates e.g., deoxyribonucleotide triphosphates. Typically, theseinclude the four standard bases, A, T, G and C. However, in certainembodiments non-natural nucleotides, such as inosine can be included. Incertain embodiments nucleotides may bear a label for detection orcapture of polynucleotides into which they are incorporated.

4. Rolling Circle Amplification

Rolling circle amplification is a method of amplifying a covalentlyclosed DNA molecule such as a single stranded, covalently closed DNAmolecule. The template DNA molecule is primed with a primer, for examplea primer provided by a primase/polymerase. A DNA polymerase performsprimer extension on the primer around the closed DNA molecule. Thepolymerase displaces the hybridized copy and continues polynucleotideextension around the template to produce a concatenated amplificationproduct.

5. Multiple Displacement Amplification

Multiple displacement amplification is an isothermal, non-PCR-based DNAamplification method in which primer extension from a template moleculeproduces molecules which themselves are primed and copied by primerextension to produce a branch-like structure. Branches are displacedfrom each other as primers are extended from one DNA molecule templateinto the branch area. In certain embodiments MDA employs random hexamersas primers to prime amplification at multiple sites on an originaltemplate and amplified copies thereof. Multiple strand amplification isfurther described in, for example, WO2011/047307A1, published Apr. 21,2011 (“Multiple Displacement Amplification”). Polymerization thatextends primers at multiple priming sites.

In certain embodiments of the disclosed methods, priming is accomplishedwith a primase/polymerase, such as TthPrimPol. In this case primingincludes the provision of deoxyribonucleotide triphosphates as areagent. In certain embodiments, the deoxyribonucleotides areunmodified. In other embodiments, deoxyribonucleotides can be modifiedby attachments to a label, for example, a fluorescent molecule. As usedherein, the term “label” refers to a chemical moiety attached to amolecule, such as a nucleic acid molecule. Detectable labels include,for example, fluorescent labels, luminescent labels, enzymatic labels,colorimetric labels such as colloidal gold or colored glass or plasticbeads and radioactive labels.

Referring to FIG. 2, in methods disclosed herein, a single stranded,covalently closed nucleic acid molecule template (“cfDNA pretreated toadd hairpin adaptors at both ends”) is provided. A primase/polymerase,such as TthPrimPol, is provided and recognizes recognition sites in thehairpin adapter and primes polymerization by the synthesis of primers.(“TthPrimPol hairpin recognition and primer synthesis”.) A DNApolymerase with preference for binding single-stranded nucleic acidmolecules and strand displacement activity amplifies the templatethrough a combination of rolling circle amplification and multipledisplacement amplification. Rolling circle amplification provides aconcatenated molecule that folds back on itself forming double-strandedsegments based on the complementary sequences. These folds also includethe adapter sequences comprising primase/polymerase recognition sites.The primase/polymerase synthesizes primers on the concatenated moleculewhich are, in turn, extended by the DNA polymerase. The result isexponential amplification of the original template molecule, typicallyin branched fashion. (“Stranded displacement and exponential rollingcircle amplification by new priming events”.)

6. Other Amplification Methods

Contemplated herein are other methods of amplifying single-stranded,covalently closed DNA molecules.

In one method, rather than priming polymerization with aprimase/polymerase, amplification is primed with random sequenceprimers. For example, random sequence primers can be hexamers comprisinga degenerate set of sequences. Amplification can continue by multipledisplacement amplification.

In another method, amplification of single-stranded, covalently closedDNA molecules is performed by Degenerate Oligonucleotide Primed(DOP)-PCR. (DOP)-PCR uses a single primer for PCR (instead of a forwardand reverse primer). This primer is usually an oligomer having about 22bases with a six-nucleotide degenerate region in the center, e.g.5′CGACTCGAGNNNNNNATGTGG 3′ [SEQ ID NO: 9]. This degenerate region is arandom sequence composed of any of the four DNA nucleotides. The firstfive steps of the DOP-PCR procedure are a non-specific amplificationstep. The degenerate primer along with low annealing temperatures willcause random annealing at locations across the entire genome. DuringPCR, extension will occur from these primers and create long fragments.

In another method, amplification of single-stranded, covalently closedDNA molecules is performed by Primer Extension Preamplification (PEP).Primer Extension Preamplification (PEP) uses random/degenerate primersand a low PCR annealing temperature. The primers can be, for example,about 15 nucleotides long.

In another method, amplification of single-stranded, covalently closedDNA molecules is performed by linker-adaptor PCR (LA-PCR). In LA-PCR,double-stranded DNA is digested with Msel, leaving a TA overhang foradapter annealing and subsequent ligation. A single primer,complementary to the adapter, is used to amplify the whole sample byPCR.

In another method, amplification of single-stranded, covalently closedDNA molecules is performed by using any combination of a thermostableDNA polymerase (e.g., Taq polymerase) and a highly processivestrand-displacement DNA polymerase (e.g., Phi29 polymerase or Bacillusstearothermophilus (Bst) polymerase). One such method is MultipleAnnealing and Looping Based Amplification Cycles (MALBAC). MALBAC is anon-exponential whole genome amplification method. Primers used inMALBAC allow amplicons to have complementary ends which form loops,inhibiting exponential copying.

D. DNA Sequencing

As used herein, the term “high throughput sequencing” refers to thesimultaneous or near simultaneous sequencing of thousands of nucleicacid molecules. High throughput sequencing is sometimes referred to as“next generation sequencing” or “massively parallel sequencing”.Platforms for high throughput sequencing include, without limitation,massively parallel signature sequencing (MPSS), Polony sequencing, 454pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, IonTorrent semiconductor sequencing, DNA nanoball sequencing, Heliscopesingle molecule sequencing, single molecule real time (SMRT) sequencing(PacBio), and nanopore DNA sequencing (e.g., Oxford Nanopore).

Methods described herein can be used for, without limitation, wholegenome sequencing, exome sequencing and amplicon sequencing. To theextent apoptotic cfDNA comprises sequences from the entire genome, theamplification product of methods described herein represent whole genomeamplification. However, amplified molecules themselves, can be subjectto amplification of specific amplicons. Sequence capture using baitsdirected to gene sequences in the genome can be used to isolateamplified molecules representing the exome. By reverse transcribing mRNAinto double stranded cDNA an amplified transcriptome can be produced forsequencing.

DNA amplified by the methods disclosed herein has properties ofdouble-stranded DNA. This is due, at least in part, to thecomplementarity within strands which fold back on themselves.Accordingly, amplified DNA can be prepared for sequencing as one mightnative double-stranded DNA. Library preparation methods depend on boththe sequencing platform and the sequencing approach. For example, thesequencing platform may be adapted for short reads, such as Illumina orIon Torrent, or for long reads, such as PacBio or Oxford Nanopore.Sequencing approaches include targeted, whole exome or whole genomesequencing. For example, to perform whole genome sequencing usingIllumina, one can shear the DNA (enzymatically or mechanically) to alength appropriate to the sequencing specifications (e.g., single-end orpaired-end reads and chosen lengths (150 nucleotides, 250 nucleotides,etc.). After shearing, the libraries are prepared using kits thatinclude the adaptors suitable for the sequencing to occur. In contrast,when using long-read whole genome sequencing, e.g., PacBio and OxfordNanopore, a first step with T7 endonuclease is recommended to eliminatethe multi-branched DNA structure derived from the multiple displacementamplification mechanism produced by all MDA methods. Otherwise,different library prep methods are followed depending on the sequencingplatform. In targeted approaches, PCR is used to amplify certain regionsof interest, that will be the only ones sequenced afterwards.

III. Kits

Also provided herein are kits for use in performing the methodsdisclosed herein. As used herein, the term “kit” refers to a collectionof items intended for use together.

Certain kits disclosed herein include 2, 3, 4, 5, 6, 7, elementsselected from: (1) a PrimPol enzyme (e.g., TthPrimPol); (2) a DNApolymerase (e.g., Phi29); (3) a single strand hairpin adaptor; (4) oneor more enzymes for dsDNA end repair (e.g., T4 DNA polymerase, klenowfragment and/or Taq polymerase); (5) one or more enzymes for DNAligation; (6) dNTPs; (7) an enhancer, e.g. to increase DNA ligationefficiency, e.g., polyethylene glycol; (8) reaction buffer and (9) abuffer for use with any of the aforementioned elements. The kits cancomprise containers to hold these reagents for. Kits can includecontainers to hold reagents. Containers, themselves, can be placed intoa shipping container. The container can be transmitted by hand deliveryor by a common carrier, such as a national postal system or a deliveryservice such as FedEx. Kits also can contain a container for shippingcollected blood to a central facility, such as a box or a bag. Kits canalso typically include instructions for use as well as and software fordata analysis and interpretation.

EXAMPLES Example 1 Hairpin Addition at Both DNA Ends According to theDisclosure Allows the Efficient Amplification of Short DNA Molecules byTruePrime Multiple Displacement Amplification

Shown in FIG. 3 is the amplification of short DNA molecules (200 bp)obtained by PCR amplification. The end-repair and dA-tailing reaction,and the addition of the hairpin adaptors enables the efficientamplification of the short DNA input by TruePrime.

The presence in DNA 1 of a single restriction recognition site for EcoRlenzyme allowed us to obtain single units of the amplified material (seeFIG. 4), demonstrating the amplification mechanism illustrated in FIG.2.

Example 2 TruePrime Rolling Circle Amplification Shows More SpecificAmplification of the Target Molecule than MDA Methods Based on RandomSynthetic Primers

Shown in FIG. 5 is the amplification of short DNA molecules (200 bp)obtained by PCR amplification and subjected to the procedure of thedisclosure: end-repair, dA-tailing, hairpin-adaptor ligation and rollingcircle amplification. The rolling circle amplification can be carriedout either with TthPrimPol (TruePrime) or random synthetic primers (RPs)to trigger the amplification, using two independently- prepared DNAsamples as substrate (Prep 1 and Prep 2). The amplification yield ismuch higher when using random primers (see FIG. 5 upper part). However,the analysis of the same amount of amplified DNA (500 ng) by restrictionenzyme digestion reveals that TruePrime (based on TthPrimPol) producesmore target molecules (236 bp) than random primers, probably due to theamplification of primer dimers, which is a well-known drawback of MDAmethods based on random synthetic primers.

Example 3 Hairpin Addition at Both DNA Ends According to the DisclosureAllows the Efficient Amplification of DNA Molecules from 50 bp up to1250 bp by TruePrime Multiple Displacement Amplification

Shown in FIG. 6 is the amplification of DNA molecules ranging from 50 bpup to 1250 bp (50, 100, 150, 200, 400, 500, 600, 700, 800 and 1250 bp)obtained by PCR amplification. The end-repair and dA-tailing reaction,and the addition of the hairpin adaptors enables the efficientamplification of the different DNA molecules by TruePrime.

Example 4 TthPrimPol DNA Primase Efficiency Depending on the HairpinSequence of the Adaptor

Shown in FIG. 7 are the different hairpin adaptors tested. All theadaptors have self-complementary sequences that allow auto-hybridizationof the molecules forming a hairpin. The hairpin sequence and lengthdiffer in each case.

Shown in FIG. 8 is the DNA primase activity of TthPrimPol, the enzyme incharge of synthesizing the DNA primers for Phi29 DNA pol in TruePrime,using adaptors with different hairpin sizes and sequences. Adaptor 1shows the highest activity, as can be deduced from the amount of DNAprimers generated, as well as the almost complete absence of theunincorporated labelled nucleotide (G*), which highlights the efficiencyof the DNA primase activity using this molecule. Surprisingly, the otherthree adaptors that contain a more accessible TthPrimPol recognitionsequence (3′ CTCC 5′)⁴⁶ show less activity, especially in the case ofadaptor 2. Adaptor 1 shows the highest activity and the shortestsequence, so it becomes the best candidate to be used in the cfDNAamplification workflow.

Example 5 Present Method Results in a Very Efficient Amplification ofCell-Free DNA Samples from Cancer Patients

48 cancer patients were recruited for this study under informed consent.10 mls of blood were extracted using Streck Cell-free DNA BCT® tubes. 3mls pf plasma were immediately isolated through a double-spincentrifugation protocol to avoid genomic DNA contamination fromnucleated blood cells. Cell-free DNA was purified from 1 ml of plasmasamples using the gold-standard cfDNA purification kit (Qiagen QlAampCirculating Nucleic Acid Kit). Different yields were obtained in eachcase quantified by Qubit (ranging from 0.12 up to 21.6 ng/μl). Cell-freeDNA size profile was analyzed using the Bioanalyzer HS kit to confirmthe presence of the apoptotic cell-free DNA molecules of interest (size-160-170 bp) and the absence of other longer DNA molecules.

1 ng of cfDNA in each case followed the disclosure workflow for cfDNAamplification (steps shown in FIG. 9, left part); i.e. beginning withthe steps of end-repair, dA-tailing and adaptor 1 ligation. After that,resulting samples (15 μl) were amplified using the TruePrime technology.Shown in FIG. 9 (right part) are the amplification yields obtained. Allcell-free DNA samples were efficiently amplified.

Example 6 Present Method Results in a Very Sensitive Amplification ofCell-Free DNA Samples from Cancer Patients

Shown in FIG. 10 are the excellent sensitivity and efficiency of thedisclosure workflow, which achieves DNA amplification yields in therange of micrograms starting from picograms of cell-free DNA. It isremarkable the proportionality between the input amount and theamplification yield observed.

Example 7 Short-Read (Illumina) and Long-Read (Oxford Nanopore MinION)Whole Genome Sequencing Confirms the Feasibility and Efficiency of theMethod Workflow for the Amplification of Apoptotic Cell-Free DNA

4 ng of cell-free DNA from a colon cancer patient (T3N1aM0) weresubjected to the disclosure workflow for cfDNA amplification induplicate.

Long-read (Oxford Nanopore MinION) whole genome sequencing: 1500 ng ofeach amplified cell-free DNA were pre-treated with T7 endonuclease Ibefore preparing the library to eliminate the multi-branched DNAstructure. Ligation ID sequencing kit SQK-LSK108 was used and protocolID genomic DNA sequencing for the MinION device using SQK-LSK 108 wasfollowed. The flow cell was run for 48 hours.

The sequences from the Oxford Nanopore MinION run were analyzed andtested for the occurrence of the hairpin adaptor sequence [SEQ ID NO:1].Although the sequencing quality was not high, the sequence of thehairpin adaptor [SEQ ID NO:1] could be found in almost every read andseparated sequence fragments with a high similarity (proven by BLAST ofthose fragments, which had identical genetic region as hit).

Shown in FIG. 11 is the high correlation of fragments separated by thehairpin adaptor sequence [SEQ ID NO:1] in respect to the length of theMinION read.

Short-read (Illumina) whole genome sequencing: 1000 ng of each amplifiedcell-free DNA were sheared with Covaris to obtain 500 bp fragments.Sheared DNA was purified using AMPure beads and the library was preparedusing the NxSeq AmpFREE Low DNA Library kit (Lucigen). Dual indices wereadded by PCR and the samples were sequenced in an Illumine HiSeq 2500using paired-end reads (2×150 bp).

Shown in FIG. 12 are the coverage and copy number variant (CNV)detection results. The coverage of two samples from the same patientlooks nearly identical and highly even, as does the CNV plot, in whichexcept for single parts both samples have an identical composition ofploidies. The reads were analyzed by CLC Genomics Workbench, combinedinto one read and then separated again at the position of the hairpin.The hairpin sequence could be found in almost all combined reads. Theagain separated and artificially created new paired reads were alignedto the human genome and analyzed for the coverage statistics,reproducibility and CNV content.

Shown in FIG. 13 is the coverage plot from the two samples from onepatient in different resolutions. It is remarkable the almost identicalamplification and results from the post-sequencing process.

Example 8 Amplicon Sequencing Confirms the Feasibility and Efficiency ofthe Method Workflow for the Amplification of Apoptotic Cell-Free DNA

26.7, 84 and 150 ng of cell-free DNA from three different colon cancerpatients (T3/4) were subjected to disclosure workflow for cfDNAamplification, obtaining 15, 17 and 20 μgs respectively. 20 ng of thenon-amplified cfDNA and 50 ng of the amplified cfDNA from each patientwere sequenced using the Oncomine™ Colon cfDNA Assay with tag molecularbarcodes for multiplexing in an Ion Proton™ sequencing system, using anIon Proton™ Chip. Libraries were prepared using the Ion Chef™ system.Read alignment was carried out using the Torrent Suite Software and thevariant calling was performed using the CLC software with the followingsettings: Ploidy=2. Ignore positions with coverage above=1000000.Restrict calling to target regions=Oncomine_Colon_cfDNA.03062017.Designed_BED. Ignore broken pairs=No. Ignore non-specific matches=Reads.Minimum coverage=10. Minimum count=2. Minimum frequency (%)=1.0−35.0.Base quality filter=No. Read direction filter=No. Relative readdirection filter=No. Read position filter=No. Remove pyro-errorvariants=No. Create track=Yes. Create annotated table=No.

Variant calling was carried out for single nucleotide variants, multiplenucleotide variants, insertions and deletions at different frequencies,from 35% to 1%. Shown in FIG. 14 are the number of annotated andnon-annotated variants detected in each patient comparing the amplifiedand non-amplified cfDNA samples in each case.

In the three cases, more variants were detected in the amplified samplethan in the non-amplified one, independently of the mutation allelefrequency threshold. Additionally, the number of annotated variants isalso higher in the amplified samples than in the non-amplified ones.

Shown in FIG. 15 are the results obtained for the first patient and thehigher number of clinically relevant variants in the amplified sample incomparison to the non-amplified cfDNA sample from the same patient attwo different allele frequencies (1% and 35%). For the 1% frequency, 30clinically relevant (ClinVar) mutations are detected in the amplifiedsample, while only 14 in the non-amplified one. From those 14, 10 arealso detected in the amplified sample. For the 35% frequency, 3 ClinVarmutations are detected in the non-amplified sample. Those 3 and 2additional ClinVar mutations are found in the amplified material.

Shown in FIG. 16 are the results obtained for the second patient and thehigher number of clinically relevant variants in the amplified sample incomparison to the non-amplified cfDNA sample from the same patient attwo different allele frequencies (1% and 35%). For the 1% frequency, 31ClinVar mutations are detected in the amplified sample, while only 13 inthe non-amplified one. From those 13, 10 are also covered in theamplified sample.

Shown in FIG. 17 are the results obtained for the third patient and thehigher number of clinically relevant variants in the amplified sample incomparison to the non-amplified cfDNA sample from the same patient attwo different allele frequencies (1% and 35%). For the 1% frequency, 26ClinVar mutations are detected in the amplified sample, while only 16 inthe non-amplified one. From those 16, 13 are also covered in theamplified sample.

Therefore, the use of the procedure of the disclosure before ampliconsequencing increases the sensitivity of the analysis, enabling thedetection of more clinically relevant variants.

EXEMPLARY EMBODIMENTS

Exemplary embodiments of the invention include the following:

1. A method of amplifying DNA comprising: a) providing linear doublestranded DNA molecules; b) attaching single-stranded adaptors to bothends of the linear double stranded DNA molecules to produce singlestranded, covalently closed DNA molecules; and c) amplifying thesingle-stranded, covalently closed DNA molecules in a single operationby (i) rolling circle amplification and (ii) multiple displacementamplification.

2. The method of embodiment 1, wherein the linear double stranded DNAmolecules comprise apoptotic cell free DNA molecules, e.g., having sizesless than 480 bp, less than 320 bp, or between about 140 bp and 180 bp(e.g., averaging about 160 bp).

3. The method of embodiment 1, wherein providing the linear doublestranded DNA comprises fragmenting chromosomal DNA.

4. The method of any of the previous embodiments wherein the lineardouble stranded DNA is derived from one or more bodily fluids frommammals, e.g., selected from CSF, blood, plasma, serum, ascites, urine,saliva, tear drops, milk, semen and synovial fluid.

5. The method of any of the previous embodiments, wherein providing thelinear double stranded DNA comprises isolating the linear doublestranded DNA from other cellular components in a biological sample.

6. The method of any of the previous embodiments, wherein providing thelinear double stranded DNA comprises end-repair and/or dA-tailing.

7. The method of embodiment 5, wherein end repair is performed using oneor more of T4 polynucleotide kinase (PNK), T4 DNA polymerase Klenowfragment and T4 DNA polymerase large fragment.

8. The method of embodiment 5, comprising performing dA tailing usingTaq polymerase.

9. The method of any of the previous embodiments, wherein the adaptorscomprise single-stranded DNA molecules.

10. The method of any of the previous embodiments, wherein the adaptorshave a hairpin structure.

11. The method of any of the previous embodiments, comprising attachingadaptors having the following sequence:5′TAACATTTGTTGGCCACTCAGGCCAACAAATGTTAT3′ [SEQ ID NO:1].

12. The method of any of the previous embodiments, wherein the adaptorscomprise a primase/polymerase recognition sequence.

13. The method of any of the previous embodiments, wherein attaching theadaptors comprises blunt-end ligation or sticky-end ligation.

14. The method of any of the previous embodiments, comprising:

providing apoptotic cell free DNA molecules;

performing end repair and dA tailing of the cell free DNA molecules; and

ligating hairpin adaptors comprising a dT overhang to the end repaired,dA tailed cell free DNA molecules.

15. The method of any of the previous embodiments, wherein amplificationis primed by a primase/polymerase.

16. The method of any of the previous embodiments, wherein amplificationis primed by Thermus thermophilus primase/polymerase (TthPrimPol).

17. The method of any of the previous embodiments, wherein amplificationis primed by a TthPrimPol having the sequence of SEQ ID NO: 10.

18. The method of any of the previous embodiments, wherein amplificationis primed by human primase/polymerase (HsPrimPol).

19. The method of any of the previous embodiments, wherein amplificationis primed with random synthetic primers (e.g., random sequencestypically 3 to 8 nucleotides in length, such as hexamers).

20. The method of any of the previous embodiments wherein amplificationcomprises strand extension using a polymerase having strand displacementactivity.

21. The method of any of the previous embodiments wherein amplificationcomprises strand extension using Phi29 polymerase.

22. The method of any of the previous embodiments wherein amplificationcomprises primase-initiated multiple displacement amplification.

23. The method of any of the previous embodiments comprising using aprimase/polymerase to generate primers on the DNA and using a polymerasehaving strand displacement activity to extend the primers.

24. The method of embodiment 23, wherein the primase/polymerasecomprises TthPrimPol and the polymerase comprises Phi29 polymerase.

25. The method of any of the previous embodiments further comprising: d)sequencing the amplified DNA.

26. The method of embodiment 25, wherein sequencing comprisesfragmenting the amplified DNA and attaching to the fragmented DNAsequencing platform-specific adaptors.

27. The method of embodiment 25, wherein sequencing is performed onselected sequences (amplicons), exomes, transcriptome or whole genome.

28. The method of embodiment 25, further comprising: e) detecting one ora plurality of genetic variants in the sequenced, amplified DNA.

29. The method of any of the preceding embodiments, further comprisingquantifying the amplified DNA.

30. A method of amplifying DNA comprising: a) providing linear doublestranded DNA molecules; b) performing end-repair and dA tailing on theDNA molecules; c) ligating hairpin adaptors to the end-repaireddA-tailed DNA molecules to produce adaptor-tagged DNA molecules; and d)amplifying the adaptor-tagged DNA molecules by (i) rolling circleamplification and (ii) multiple displacement amplification (“MDA”).

31. The method of embodiment 30, wherein amplification comprisesrandom-primed MDA, using Phi29 DNA polymerase and random syntheticprimers.

32. The method of embodiment 30, wherein amplification comprises primingwith a primase/polymerase (e.g., TthPrimPol).

33. A method of amplifying DNA comprising: a) providing linear doublestranded DNA molecules; b) attaching single-stranded adaptors to bothends of the linear double stranded DNA molecules to produce singlestranded, covalently closed DNA molecules; and c) amplifying thesingle-stranded, covalently closed DNA molecules by any combination of athermostable DNA polymerase, e.g., Taq polymerase, random or degenerateprimers and an optional ligase.

34. The method of embodiment 33, wherein amplification comprisesDegenerate oligonucleotide-primed PCR (DOP-PCR), linker-adaptor PCR(LA-PCR), Primer Extension Pre-amplification PCR (PEP-PCR-/I-PEP-PCR),and variations thereof).

35. A method of amplifying DNA comprising: a) providing linear doublestranded DNA molecules; b) attaching single-stranded adaptors to bothends of the linear double stranded DNA molecules to produce singlestranded, covalently closed DNA molecules; and c) amplifying thesingle-stranded, covalently closed DNA molecules using any combinationof a thermostable DNA polymerase (e.g., Taq polymerase) and a highlyprocessive strand-displacement DNA polymerase (e.g., Phi29 polymerase orBacillus stearothermophilus (Bst) polymerase).

36. The method of embodiment 35, wherein amplification comprisesmultiple annealing and looping-based amplification cycles (MALBAC).

37. A kit comprising: (a) a primase/polymerase;

(b) a polymerase having strand displacement activity; (c)deoxyribonucleotide triphosphates and (d) a single stranded hairpinadaptor.

38. The kit of embodiment 37, wherein the primase/polymerase isTthPrimPol.

39. The kit of embodiment 37, wherein the polymerase is Phi29 DNApolymerase.

40. The kit of embodiment 37, wherein the single strand adaptor has thesequence: 5′ TAACATTTGTTGGCCACTCAGGCCAACAAATGTTAT 3′ [SEQ ID NO: 1].

41. The kit of embodiment 37, wherein the kit further comprises: d) one,two or three elements selected from: (i) one or more enzymes for dsDNAend repair (e.g., T4 DNA polymerase, klenow fragment and/or Taqpolymerase); (ii) one or more enzymes for DNA ligation; (iii)optionally, a reagent to increase DNA ligation efficiency; (iv) reactionbuffer; and (v) a buffer for use with any of the aforementionedelements.

42. The kit of any of the preceding embodiments wherein the kit furthercomprises: (a) reagents for isolation of apoptotic cell free DNA.

43. A DNA library comprising a population of adapter tagged,single-stranded, covalently closed DNA molecules, wherein each DNAmolecule comprises first, second, third and fourth regions, wherein thesecond and fourth regions are complementary to each other and the firstand third regions are not complementary to each other.

44. The DNA library of embodiment 43, wherein, the first and thirdregions have identical sequences.

45. The DNA library of embodiment 43 or embodiment 44, wherein thesecond and fourth regions comprise a segment of genomic DNA, e.g.,apoptotic cfDNA.

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As used herein, the following meanings apply unless otherwise specified.The word “may” is used in a permissive sense (i.e., meaning having thepotential to), rather than the mandatory sense (i.e., meaning must). Thewords “include”, “including”, and “includes” and the like meanincluding, but not limited to. The singular forms “a,” “an,” and “the”include plural referents. Thus, for example, reference to “an element”includes a combination of two or more elements, notwithstanding use ofother terms and phrases for one or more elements, such as “one or more.”The term “or” is, unless indicated otherwise, non-exclusive, i.e.,encompassing both “and” and “or.” The term “any of” between a modifierand a sequence means that the modifier modifies each member of thesequence. So, for example, the phrase “at least any of 1, 2 or 3” means“at least 1, at least 2 or at least 3”.

It should be understood that the description and the drawings are notintended to limit the invention to the particular form disclosed, but tothe contrary, the intention is to cover all modifications, equivalents,and alternatives falling within the spirit and scope of the presentinvention as defined by the appended claims. Further modifications andalternative embodiments of various aspects of the invention will beapparent to those skilled in the art in view of this description.Accordingly, this description and the drawings are to be construed asillustrative only and are for the purpose of teaching those skilled inthe art the general manner of carrying out the invention. It is to beunderstood that the forms of the invention shown and described hereinare to be taken as examples of embodiments. Elements and materials maybe substituted for those illustrated and described herein, parts andprocesses may be reversed or omitted, and certain features of theinvention may be utilized independently, all as would be apparent to oneskilled in the art after having the benefit of this description of theinvention. Changes may be made in the elements described herein withoutdeparting from the spirit and scope of the invention as described in thefollowing claims. Headings used herein are for organizational purposesonly and are not meant to be used to limit the scope of the description.

What is claimed is:
 1. A method of amplifying DNA comprising: a)providing linear double stranded DNA molecules; b) attachingsingle-stranded adaptors to both ends of the linear double stranded DNAmolecules to produce single stranded, covalently closed DNA molecules;and c) amplifying the single-stranded, covalently closed DNA moleculesin a single operation by (i) rolling circle amplification and (ii)multiple displacement amplification.
 2. The method of claim 1, whereinthe linear double stranded DNA molecules comprise apoptotic cell freeDNA molecules, e.g., having sizes less than 480 bp, less than 320 bp, orbetween about 140 bp and 180 bp (e.g., averaging about 160 bp).
 3. Themethod of claim 1, wherein providing the linear double stranded DNAcomprises fragmenting chromosomal DNA.
 4. The method of any of theprevious claims wherein the linear double stranded DNA is derived fromone or more bodily fluids from mammals, e.g., selected from CSF, blood,plasma, serum, ascites, urine, saliva, tear drops, milk, semen andsynovial fluid.
 5. The method of any of the previous claims, whereinproviding the linear double stranded DNA comprises isolating the lineardouble stranded DNA from other cellular components in a biologicalsample.
 6. The method of any of the previous claims, wherein providingthe linear double stranded DNA comprises end-repair and/or dA-tailing.7. The method of claim 5, wherein end repair is performed using one ormore of T4 polynucleotide kinase (PNK), T4 DNA polymerase Klenowfragment and T4 DNA polymerase large fragment.
 8. The method of claim 5,comprising performing dA tailing using Taq polymerase.
 9. The method ofany of the previous claims, wherein the adaptors comprisesingle-stranded DNA molecules.
 10. The method of any of the previousclaims, wherein the adaptors have a hairpin structure.
 11. The method ofany of the previous claims, comprising attaching adaptors having thefollowing sequence: 5′TAACATTTGTTGGCCACTCAGGCCAACAAATGTTAT3′ [SEQ IDNO:1].
 12. The method of any of the previous claims, wherein theadaptors comprise a primase/polymerase recognition sequence.
 13. Themethod of any of the previous claims, wherein attaching the adaptorscomprises blunt-end ligation or sticky-end ligation.
 14. The method ofany of the previous claims, comprising: providing apoptotic cell freeDNA molecules; performing end repair and dA tailing of the cell free DNAmolecules; and ligating hairpin adaptors comprising a dT overhang to theend repaired, dA tailed cell free DNA molecules.
 15. The method of anyof the previous claims, wherein amplification is primed by aprimase/polymerase.
 16. The method of any of the previous claims,wherein amplification is primed by Thermus thermophilusprimase/polymerase (TthPrimPol).
 17. The method of any of the previousclaims, wherein amplification is primed by a TthPrimPol having thesequence of SEQ ID NO:
 10. 18. The method of any of the previous claims,wherein amplification is primed by human primase/polymerase (HsPrimPol).19. The method of any of the previous claims, wherein amplification isprimed with random synthetic primers (e.g., random hexamer sequences).20. The method of any of the previous claims wherein amplificationcomprises strand extension using a polymerase having strand displacementactivity.
 21. The method of any of the previous claims whereinamplification comprises strand extension using Phi29 polymerase.
 22. Themethod of any of the previous claims wherein amplification comprisesprimase-initiated multiple displacement amplification.
 23. The method ofany of the previous claims comprising using a primase/polymerase togenerate primers on the DNA and using a polymerase having stranddisplacement activity to extend the primers.
 24. The method of claim 23,wherein the primase/polymerase comprises TthPrimPol and the polymerasecomprises Phi29 polymerase.
 25. The method of any of the previous claimsfurther comprising: d) sequencing the amplified DNA.
 26. The method ofclaim 25, wherein sequencing comprises fragmenting the amplified DNA andattaching to the fragmented DNA sequencing platform-specific adaptors.27. The method of claim 25, wherein sequencing is performed on selectedsequences (amplicons), exomes, transcriptome or whole genome.
 28. Themethod of claim 25, further comprising: e) detecting one or a pluralityof genetic variants in the sequenced, amplified DNA.
 29. The method ofany of the preceding claims, further comprising quantifying theamplified DNA.
 30. A method of amplifying DNA comprising: a) providinglinear double stranded DNA molecules; b) performing end-repair and dAtailing on the DNA molecules; c) ligating hairpin adaptors to theend-repaired dA-tailed DNA molecules to produce adaptor-tagged DNAmolecules; and d) amplifying the adaptor-tagged DNA molecules by (i)rolling circle amplification and (ii) multiple displacementamplification (“MDA”).
 31. The method of claim 30, wherein amplificationcomprises random-primed MDA, using Phi29 DNA polymerase and randomsynthetic primers.
 32. The method of claim 30, wherein amplificationcomprises priming with a primase/polymerase (e.g., TthPrimPol).
 33. Amethod of amplifying DNA comprising: a) providing linear double strandedDNA molecules; b) attaching single-stranded adaptors to both ends of thelinear double stranded DNA molecules to produce single stranded,covalently closed DNA molecules; and c) amplifying the single-stranded,covalently closed DNA molecules by any combination of a thermostable DNApolymerase, e.g., Taq polymerase, random or degenerate primers and anoptional ligase.
 34. The method of claim 33, wherein amplificationcomprises Degenerate oligonucleotide-primed PCR (DOP-PCR),linker-adaptor PCR (LA-PCR), Primer Extension Pre-amplification PCR(PEP-PCR-I-PEP-PCR), and variations thereof).
 35. A method of amplifyingDNA comprising: a) providing linear double stranded DNA molecules; b)attaching single-stranded adaptors to both ends of the linear doublestranded DNA molecules to produce single stranded, covalently closed DNAmolecules; and c) amplifying the single-stranded, covalently closed DNAmolecules using any combination of a thermostable DNA polymerase (e.g.,Taq polymerase) and a highly processive strand-displacement DNApolymerase (e.g., Phi29 polymerase or Bacillus stearothermophilus (Bst)polymerase).
 36. The method of claim 35, wherein amplification comprisesmultiple annealing and looping-based amplification cycles (MALBAC). 37.A kit comprising: (a) a primase/polymerase; (b) a polymerase havingstrand displacement activity; (c) deoxyribonucleotide triphosphates and(d) a single stranded hairpin adaptor.
 38. The kit of claim 37, whereinthe primase/polymerase is TthPrimPol.
 39. The kit of claim 37, whereinthe polymerase is Phi29 DNA polymerase.
 40. The kit of claim 37, whereinthe single strand adaptor has the sequence: 5′TAACATTTGTTGGCCACTCAGGCCAACAAATGTTAT 3′ [SEQ ID NO: 1].
 41. The kit ofclaim 37, wherein the kit further comprises: d) one, two or threeelements selected from: (i) one or more enzymes for dsDNA end repair(e.g., T4 DNA polymerase, klenow fragment and/or Taq polymerase); (ii)one or more enzymes for DNA ligation; (iii) optionally, a reagent toincrease DNA ligation efficiency; (iv) reaction buffer; and (v) a bufferfor use with any of the aforementioned elements.
 42. The kit of any ofthe preceding claims wherein the kit further comprises: (a) reagents forisolation of apoptotic cell free DNA.
 43. A DNA library comprising apopulation of adapter tagged, single-stranded, covalently closed DNAmolecules, wherein each DNA molecule comprises first, second, third andfourth regions, wherein the second and fourth regions are complementaryto each other and the first and third regions are not complementary toeach other.
 44. The DNA library of claim 43, wherein, the first andthird regions have identical sequences.
 45. The DNA library of claim 43or claim 44, wherein the second and fourth regions comprise a segment ofgenomic DNA, e.g., apoptotic cfDNA.